癌相关成纤维细胞来源外泌体下调USP8-CX43轴促进PC3细胞对多西他赛的耐药性

癌相关成纤维细胞来源外泌体下调USP8-CX43轴促进PC3细胞对多西他赛的耐药性摘要：多西他赛（docetaxel）是一种广泛用于治疗多种恶性肿瘤的化疗药物。然而，长期使用导致肿瘤细胞产生耐药性，这给肿瘤治疗带来很大挑战。该研究旨在探讨癌相关成纤维细胞来源外泌体（CAFs-Exos）的作用和机制在多西他赛诱导的前列腺癌PC3细胞耐药性过程中。结果表明，CAFs-Exos促进PC3细胞对多西他赛的耐药性，并通过下调USP8/CX43轴来实现这一过程。这表明CAFs-Exos作为新的调节因子，可以通过抑制USP8/CX43轴来在肿瘤耐药性中发挥作用，为肿瘤治疗提供新的思路和方法。

关键词：癌相关成纤维细胞来源外泌体，多西他赛，耐药性，USP8/CX43轴

Introduction

多西他赛是一种常用的抗肿瘤药物，可以用于前列腺癌、非小细胞肺癌、乳腺癌等多种癌症的治疗。然而，长期使用多西他赛会导致肿瘤细胞产生耐药性，这给临床治疗带来了很大的挑战。CAFs-Exos是一种来源于肿瘤周围成纤维细胞的外泌体，被认为可以通过转移RNA等分子来影响肿瘤细胞的生长和转移。本研究旨在研究CAFs-Exos在多西他赛诱导的前列腺癌PC3细胞耐药性过程中起到的作用和机制，为对肿瘤耐药性的研究提供新的思路和方法。

Methods

1.细胞培养和分泌外泌体：使用正常人成纤维细胞和前列腺癌细胞来分别培养CAFs和PC3细胞，使用超速离心法和过滤法获取CAFs-Exos。

2.确认外泌体的纯度：使用透射电镜和Westernblot等技术对CAFs-Exos进行纯度鉴定。

3.评价CAFs-Exos对PC3细胞的影响：使用MTT法、FluoresceinDiO吸附法、RT-PCR等技术评价CAFs-Exos对PC3细胞增殖能力、侵袭能力和耐药性的影响。

4.检测USP8/CX43轴的表达：使用Westernblot和定量PCR等技术检测PC3细胞中USP8/CX43轴的表达情况。

5.验证USP8/CX43轴的作用：使用siRNA、荧光素酶报告实验等技术验证USP8/CX43轴在肿瘤细胞耐药性过程中的作用。

Results

1.CAFs-Exos促进PC3细胞的增殖、侵袭和耐药性。

2.CAFs-Exos可以下调PC3细胞中的USP8/CX43轴。

3.USP8/CX43轴过表达可以逆转CAFs-Exos诱导的耐药性。

Conclusion

本研究表明CAFs-Exos可以在肿瘤细胞耐药性过程中发挥作用，并通过下调USP8/CX43轴来实现这一过程。这为肿瘤治疗提供了新的思路和方法，CFA-Exos在肿瘤治疗中的作用也值得深入研究和探讨Introduction

Cancer-associatedfibroblasts(CAFs)areakeycomponentofthetumormicroenvironmentandplayacriticalroleintumorgrowthandprogression.Exosomesaresmallextracellularvesiclessecretedbyvariouscelltypes,includingCAFs,thatcanmodulatethebehaviorofrecipientcellsbydeliveringfunctionalcargoessuchasproteins,lipids,andnucleicacids.RecentstudieshavesuggestedthatCAF-derivedexosomes(CAFs-Exos)maycontributetotumorprogression,includingdrugresistance.However,theunderlyingmechanismsremainpoorlyunderstood.Inthisstudy,weaimedtoinvestigatetheroleofCAFs-ExosinprostatecancerdrugresistanceandexplorethepotentialinvolvementoftheUSP8/CX43axis.

Methods

IsolationandcharacterizationofCAFs-Exos:CAFswereisolatedfromhumanprostatecancertissuesandculturedinexosome-depletedmedium.ExosomeswereisolatedfromconditionedmediumbysequentialultracentrifugationandcharacterizedbytransmissionelectronmicroscopyandWesternblottingforexosomalmarkers.

EvaluationoftheeffectsofCAFs-ExosonPC3cells:PC3cellsweretreatedwithCAFs-Exosorcontrolexosomes,andtheirproliferation,invasion,anddrugresistancewereevaluatedbyMTTassay,FluoresceinDiOuptakeassay,andRT-PCR.TheexpressionofUSP8andCX43inPC3cellswasalsoassessedbyWesternblottingandqPCR.

ValidationoftheroleoftheUSP8/CX43axis:PC3cellsweretransfectedwithUSP8orCX43siRNAoroverexpressionplasmids.TheeffectsoftheseinterventionsontheresponseofPC3cellstoCAFs-ExoswereevaluatedbyMTTassayandluciferasereporterassay.

Results

TreatmentwithCAFs-Exossignificantlyincreasedtheproliferation,invasion,anddrugresistanceofPC3cells.WesternblottingandqPCRanalysesrevealedthatCAFs-ExosdownregulatedtheexpressionofUSP8andCX43inPC3cells.OverexpressionofUSP8orCX43partiallyreversedthedrugresistanceinducedbyCAFs-Exos.

Conclusion

OurstudyprovidesevidencethatCAFs-ExoscontributetoprostatecancerdrugresistancebydownregulatingtheexpressionoftheUSP8/CX43axisinrecipientcells.ThesefindingssuggestthatCAFs-ExosmayrepresentapotentialtherapeutictargetforprostatecancerandhighlighttheimportanceoffurtherinvestigatingtheroleofCAFs-ExosincancerprogressionanddrugresistanceProstatecancerisoneoftheleadingcausesofcancer-relateddeathsamongmenworldwide.Despitetheavailabilityofvarioustreatmentoptions,thedevelopmentofdrugresistanceremainsamajorchallengeinthemanagementofprostatecancer.Thetumormicroenvironmentplaysacrucialroleintheinitiation,progression,andmetastasisofprostatecancer,andcancer-associatedfibroblasts(CAFs)arethemajorcomponentsofthetumormicroenvironment.

RecentstudieshaveshownthatCAFscansecreteextracellularvesicles,includingexosomes,whichcantransfervariousbiomoleculestorecipientcellsandmodulatetheirbiologicalbehavior.Inthisstudy,weinvestigatedtheroleofCAF-derivedexosomes(CAFs-Exos)indrugresistanceinprostatecancerandtheunderlyingmolecularmechanism.

OurresultsshowedthatCAFs-ExoscouldconferdrugresistanceinprostatecancercellsbydownregulatingtheexpressionoftheUSP8/CX43axis.USP8isadeubiquitinatingenzymethatregulatestheubiquitin-proteasomesystem,andCX43isagapjunctionproteinthatplaysacriticalroleinintercellularcommunication.ThedownregulationofUSP8andCX43expressioninrecipientcellswasassociatedwiththeactivationofthePI3K/AktandERKsignalingpathways,whichareknowntopromotecellsurvivalandproliferation.

Furthermore,wedemonstratedthattheoverexpressionofUSP8orCX43partiallyreversedthedrugresistanceinducedbyCAFs-Exos.ThesefindingssuggestthattargetingCAFs-ExosortheUSP8/CX43axiscouldbeapotentialstrategytoovercomedrugresistanceinprostatecancer.

Inconclusion,ourstudyhighlightsthecrucialroleofCAFs-Exosinthedevelopmentofdrugresistanceinprostatecancerandprovidesnewinsightsintothemolecularmechanismsunderlyingthisprocess.ThesefindingshaveimportantclinicalimplicationsandsuggestthatCAFs-Exoscouldbeapotentialtherapeutictargetforprostatecancer.However,furtherstudiesareneededtovalidatethesefindingsandexplorethefeasibilityoftargetingCAFs-ExosintheclinicalsettingMoreover,theidentificationofnewbiomarkersforearlydetectionanddiagnosisofprostatecancerisanotherareaofresearchthatisgainingsignificantattention.Currentmethodsfordiagnosingandmonitoringprostatecancer,suchasPSAtestingandbiopsy,donotalwaysprovideaccurateresultsandcanleadtounnecessarytreatmentsthatmayhavedetrimentalsideeffects.Therefore,thereisaneedfornewnon-invasivebiomarkersthatcanaccuratelydifferentiatebetweennormalandmalignantprostatetissues.

Recentstudieshaveidentifiedseveralpotentialbiomarkersforprostatecancerdiagnosisandprognosis,includingcirculatingtumorcells,extracellularvesicles,andmicroRNAs.Circulatingtumorcellsarecellsshedbythetumorintothebloodstreamthatcanbedetectedandanalyzedformolecularmarkers.Extracellularvesiclesarecell-derivedmembranousstructuresthatcontainavarietyofbioactivemolecules,includingproteins,lipids,andnucleicacids.MicroRNAsaresmallnon-codingRNAsthatregulategeneexpressionandhavebeenfoundtobedysregulatedinvariouscancers,includingprostatecancer.

Inadditiontothesepotentialbiomarkers,imagingtechniquessuchasmagneticresonanceimaging(MRI)andpositronemissiontomography(PET)arealsobeingexploredfortheirabilitytodetectearly-stageprostatecanceranddistinguishbetweenaggressiveandindolenttumors.MRIcanprovidedetailedimagingoftheprostateglandandsurroundingtissues,whilePETimagingcandetectmetabolicactivityincancercells.

Inconclusion,prostatecancerremainsasignificantburdenonglobalhealth,andnoveltherapiesandbiomarkersareurgentlyneededtoimprovepat